Investigations concerning the morphogenesis of plant tissue in culture date back at least to the 1950's (Skoog, F. and Miller C. O. Symp Soc. Exp. Biol., 11:118 (1957)) and have continued apace to date. Several monographs provide extensive reviews of the field and contain compilations of numbers of species which will undergo plant regeneration in culture (See for example, Murashige T., In: "Propagation of Higher Plants through Tissue Culture," T. A. Thorpe, Ed., p. 15, Univ. Calgary Press, Calgary (1978); Vasil, I,K, et al Adv. Gent. 20:127 (1979) and Evans. D. A., et al In: "Plant Tissue Culture: Methods and Applications in Agriculture: T. A. Thorpe, Ed. pg. 45, Academic Press, New York (1981)).
The term "plant tissue culture" as used herein is taken in its broadest meaning to refer to the cultivation, in vitro, of all plants parts, whether a single cell, a tissue or an organ, under aseptic conditions. More restrictive terms relating to plant tissue culture technology include: "callus culture" by which is meant, the culture of cell masses on agar medium and produced from an explant of a seedling or other plant source; "cell culture" by which is meant, the culture of cells in liquid media in vessels which are usually aerated by agitation; "organ culture" by which is meant, the aseptic culture on nutrient media of embryos anthers (microospores), ovaries, roots, shoots, or other plant organs; "meristem culture and morphogenesis" by which is meant, the aseptic culture of shoot meristems or other explant tissue on nutrient media for the purpose of growing complete plants, and "protoplast culture" by which is meant, the aseptic isolation and culture of plant protoplasts from cultured cells or plant tissue.
On their face, the principles underlying plant tissue culture are quite simple. Initially, it is necessary to isolate a plant part from the intact plant and disrupt its organ, inter-tissue, and inter-cellular relationships. Subsequently, it is necessary to provide the isolated material with the appropriate environment in which to express its intrinsic or induced developmental potential. Finally, the steps must be carried out aseptically. Although the principles may be simply stated, as a matter of practice, the successful culture of plant tissue and its regeneration into a mature plant is extremely complex. The impressive list of plants species cited herein below, for which successful regeneration has been achieved, belies the difficulties in achieving those results. As will be noted later, successful regeneration of a particular species is often characterized by the addition of (or even omission of) catalytic amounts of auxins, cytokinins, or other growth regulators. Further, successful regeneration may also be a function of not only the mere presence of a certain compound but its ratio to other media components as well. Since each plant species appears to possess a relatively unique optimal set of media requirements, the successful preparation and regeneration of a new species cannot be necessarily inferred from the successful regimens applied to unrelated plant varities. This is not to say that broad generalizations as to procedure are not possible. For example, if the goal of the tissue culture system is vegetative propagation, then the regeneration may be envisioned to comprise three stages. The first stage occurs following the transfer of an explant onto a culture medium. This stage is characterized by a proliferation of the explant or callus. The second stage is characterized by a rapid increase in organ growth. This stage may require a transfer to a second medium with or without a change in growth regulator concentration. The final stage occurs when the plants are removed from in vitro culture and requires the establishment of the autotrophic state.
As mentioned previously, organogenesis or embryogenesis has been reported for a variety of species. Plant regeneration has been achieved from explants of cotyledon, hypocotyl, stems, leaf, shoot apex, root, young infloresences, flower petals, petioles, ovular tissue and embryos. For a particular species the source of the explant may be important for the success of the subsequent regeneration. The size and the shape of the explant may also be critical. Another element to be considered is the method of providing aseptic explant material for purpose of callus formation. This involves sterilization of the explant tissue prior to inoculation onto propagation medium. Even this apparently routine process is subject to a wide variety of critical experimental parameters. To illustrate this point Table I shows the extreme variability in experimental protocols for the sterilization procedures alone.
TABLE I __________________________________________________________________________ Explant Plant Size Sterilization protocol Remarks __________________________________________________________________________ Leaf blade tomato 6 .times. 8 mm wash in detergent, 10 young min. in 7% Clorox.sup.a, leaves near wash twice in water shoot apex Stem rape 5 mm rinse in 70% ethanol, basal end in 6 min. in 10% Clorox, contact with wash four times in medium water Embryo cacao 2.5-25 mm 15 min in 10% Clorox use pods intact with 0.1% Tween 20 larger than 12 cm Storage organ artichoke 2.4 .times. 2 mm 30 min in 20% Clorox, use storage wash repeatedly parenchyma region Seed (root) petunia 3 mm 30 min. 50% Clorox, use 4-6 days wash three times in after germi- water nation Seed (hypocotyl) flax 4-8 mm 1 min. in 70% ethanol, use 5 days 20 min. in 20% Clorox, after germi- wash in water nation __________________________________________________________________________ .sup. a Clorox .RTM. , a commercial bleach, is a 5% solution of sodium hypochlorite. After: Evans et al supra
Once the explant is placed onto a suitable medium callus formation may occur. Only a small percentage of the cells from an explant will give rise to the callus. A variety of factors have been reported to effect callus proliferation; medium composition, size and shape of the original explant, friability of cells of the callus and even the season of the year. The latter factor likely a reflection of changes in endogenous levels of growth regulating substances.
Somatic embryogenesis can then proceed directly from either a population of sporophytic or gametophytic cells or alternatively from embryogenic cells obtained from epigenetic redetermination of callus cells. Regardless of the mode of embryogenesis, the manipulation of growth regulators are extremely important. This fact is illustrated in Table II.
TABLE II __________________________________________________________________________ Growth Regulators Crop Species 1.degree. Medium 2.degree. Medium Medium Explant __________________________________________________________________________ Anise Pimpinella 5 .mu.M 2,4-D none B5 hypocoytl anism Asparagus Asparagus 5.4 .mu.M NAA 0.5-5.7 .mu.M IAA LS or cladodes officinalis 4.7 .mu.M KIN 0.4-17.7 .mu.M 6BA MS shoots Cacao Theobroma none 6.4 .mu.M NAA MS imm. embryo cacoa.sup.a 10% CW 10% CW cotyledon Cauliflower Brassica 5.7 .mu.M IAA 5.7 .mu.M IAA MS leaf oleracea 2.0 .mu.M KIN Caraway Carum carvi 10.7 .mu.M NAA none MS petiole Carrot Dacus carota 4.5 .mu.M 2,4-D none MS storage root Celery Apium graveolens 2.2 .mu.M 2,4-D 2.7 .mu.M KIN MS petiole Coffee Coffea arabica 18.4 .mu.M KIN 2.3 .mu.M KIN MS leaf 4.5 .mu.M 2,4-D 0.27 .mu.M NAA Coffee Coffea canephora 2 .mu.M KIN 2.5 .mu.M KIN MS leaf Coriander Coriandrum 10.7 .mu.M NAA none MS embryo sativum Cotton Gossypium 0.5 .mu.M 2,4-D 11.4 .mu.M IAA MS hypocotyl klotzschianum 4.7 .mu.M KIN Date Palm Phoenix 452 .mu.M 2,4-D none MS ovule dactylifera 4.9 .mu.M 2iP Dill Anethum 10.7 .mu.M NAA none MS embryo graveolens 2.3 .mu.M 2,4-D none White inflores- 2.3 .mu.M KIN ence Eggplant Solanum 5 .mu.M NOA 4.7 .mu.M KIN MS hypocotyl melongena Fennel Foeniculum 27.6 .mu.M 2,4-D none Nitsch stem vulgare 1 .mu.M KIN Garlic Allium sativa 10 .mu.M CPA 10 .mu.M IAA AZ stem 2 .mu.M 2,4-D 20 .mu.M KIN 0.5 .mu.M KIN Ginseng Panax ginseng 2.2 .mu.M 2,4-D 0.4 .mu.M 2,4-D MS pith 0.8 .mu.M KIN Grapes Vitis spp. 4.5 .mu.M 2,4-D 10.7 .mu.M NAA MS flower, 0.4 .mu.M 6BA 0.4 .mu.M 6BA leaf Oil Palm Elaeis 4.5 .mu.M 2,4-D 5.7 .mu.M IAA Heller embryo guineensis 2.3 .mu.M KIN Orange Citrus 0.5 .mu.M KIN none Murashige, ovule sinensis 5.7 .mu.M IAA Tucker Parsley Petroselinum 27 .mu.M 2,4-D none Hilde- petiole hortense brandt C Pumpkin Cucurbita pepo 4.9 .mu.M IBA 1.4 .mu.M 2,4-D MS cotyledon hypocotyl Sandal- Santalum album 9.1 .mu.M 2,4-D none White embryo wood 23.2 .mu.M KIN S. album 4.5 .mu.M 2,4-D 1.5-5.8 .mu.M GA.sub.3 MS stem 0.9-2.3 .mu.M KIN White Sweet Gum Liquidambar 5.3 .mu.M NAA none Blaydes hypocotyl styraciflua 8.8 .mu.M 6BA Water Sium suave 10.7 .mu.M NAA none MS embryo Parsnip Papaya Carica papaya 1 .mu.M NAA 0.1 .mu.M NASA White petiole 10 .mu.M 2iP 0.01 .mu.M 6BA __________________________________________________________________________ .sup.a Requires a subculture on a maintenance medium prior to 2.degree. culture. After: Evans et al supra Abbreviations auxins: IAA = indole acetic acid; IBA = indole butyric acid; 2,4D = 2,4dichlorophenoxyacetic acid; NAA = naphthaleneacetic acid; pCPA = parachlorophenoxyacetic acid; NOA = Bnapthoxyacetic acid; BTOA = 2benzothiazole acetic acid; PIC = picloram; 2,4,5T = 2,4,5tichlorophenoxyacetic acid; cytokinins: KIN = kinetin; 6BA = 6 benzyladenine (benzylaminopurine) 2iP = 2 isopentenyl adenine; ZEA = zeatin; other growth regulators: ADE = adenine; CW = coconut water; CH = casein hydrolysate; ABA = abscisic acid; GA.sub.3 = gibberellic acid. Medium MS = Murashige and Skoog B5 = Gamborg SH = Schenk and Hildebrandt W = White LS = Linsmaier and Skoog
Even though cotton is listed among those species for which embryogenesis had occurred, the development of the embryoids into plants was not achieved. The cotton species used in those experiments was not a cultivated cotton of commercial value but a lintless wild cotton.
It is the object of this invention to provide a method for the controlled regeneration of cotton plants from tissue culture.